Project Summary/Abstract Carbohydrates comprise one of the largest, most diverse collections of biologically active molecules. However, relative to other biomolecules such as nucleic acids and proteins, carbohydrates remain poorly understood due to challenges in their detection, synthesis, and analysis. The broad objective of this program is to develop chemical approaches to advance a fundamental understanding of the roles of carbohydrates in biology and disease. In the last granting period, we developed a novel Networking of OGT Interactors and Substrates (NOIS) method to study the biological functions of O-linked ?-N-acetylglucosamine (O-GlcNAc) glycosylation. O-GlcNAc is an abundant, essential post-translational modification that is emerging as a key regulator of many physiological functions, ranging from epigenetic and transcriptional gene regulation to insulin signaling, cancer cell metabolism, and neurodegeneration. Our NOIS approach combines new chemoproteomic tools, genetic engineering, MS analysis, and bioinformatics methods to examine interconnections between the interactors and substrates of O-GlcNAc transferase (OGT) across the proteome both in vitro and in vivo. These analyses have revealed novel, unexpected functions for the O-GlcNAc modification and highlighted potential mechanisms to explain the unique specificity of OGT. In the coming granting period, we will expand this approach to important physiological and disease contexts and tackle the next set of critical barriers in the field. In Aim 1, we will develop next-generation chemoproteomic tools and MS methodologies to quantify changes in O-GlcNAc networks in response to biological stimuli. In Aim 2, we will apply our quantitative NOIS method to study the dynamics of O-GlcNAc networks during both normal and excitotoxic neuronal stimulation, and the network perturbations in an in vivo model of late-onset Alzheimer's disease (AD) and metabolic syndrome/T2DM-associated dementia. In Aim 3, we will demonstrate how NOIS can produce novel, tractable hypotheses and test those hypotheses to establish previously undiscovered functions for O-GlcNAc in synaptic plasticity. We will also use NOIS to explore ways to selectively modulate OGT's activity toward specific substrates. Finally, in Aim 4, we will apply similar proteomic and bioinformatics approaches to investigate the crosstalk between O-GlcNAc and phosphorylation, as well as its impact on neuronal signaling pathways critical for neuronal communication, homeostasis, and synaptic plasticity. Together, the proposed studies will provide a unified approach to track dynamic O-GlcNAcylation events across the proteome and identify physiologically important and/or disease-causing O-GlcNAcylation events. In turn, this information should provide new potential therapeutic targets or approaches to combat progressive neurodegeneration, Alzheimer's disease, and related dementias.